Methods and probes for identifying a nucleotide sequence

ABSTRACT

The present invention provides a method for identifying a set of target nucleotide sequences capable of identifying a member of a group of related nucleotide sequences, the method comprising the step of dividing the nucleotide sequence of each member of the group into a plurality of subsequences, wherein at least two of the subsequences overlap. The method is useful in generating probe sets capable of assigning alleles at HLA or KIR loci.

FIELD OF THE INVENTION

The present invention is directed to the field of molecular biology. More specifically the invention is directed to methods for generating oligonucleotide probes and uses thereof in identifying members of a group of related nucleotide sequences. The methods and probes may be used in identifying an allele of a gene in an individual.

BACKGROUND TO THE INVENTION

The Human Genome Project has highlighted the importance of single nucleotide polymorphisms (SNPs) in the genome. These polymorphisms occur on average every 100 to 300 bases throughout the genome. While the genes of all humans are known to be more than 99% identical, it is presence of SNPs that provide a major component of genetic diversity in a species. Different alleles of a gene can confer very different phenotypes on an individual including characteristics as diverse as disease resistance, the ability to respond to a pharmaceutical compound, sporting ability and the like.

Plant genomes also contain SNPs that can result in different characteristics. SNPs are increasingly becoming the marker of choice in genetic analysis and are used routinely as markers in agricultural breeding programs. SNPs cannot only be used to link a particular genotype to phenotype. They can also be used as a “fingerprint” in identifying organisms as diverse as bacteria, viruses and the like.

The ability to ascribe a genotype to an individual is of significance for a number of reasons. As a broad concept this involves identification of a nucleotide sequence of a subject gene of the organism involved. The most direct manner of providing this information is to sequence the subject gene. While automated sequencing has been possible for some years, the process is still time intensive and expensive.

As a result of the limitations to the widespread use of direct sequencing, a number of indirect methods have been advanced to identify alleles. One of the simplest is the use of Restriction Fragment Length Polymorphism (RFLP). This approach relies on the specificity of restriction endonucleases for certain nucleotide sequences. Thus, if a certain sequence is present, the endonuclease will cleave the polynucleotide, and if not no cleavage will result. Different genotypes are detected by the different pattern of restriction fragments, as detected by gel electrophoresis. The disadvantage of this method is that where there is no endonuclease specific for each and every SNP in the range of alleles, then all alleles will not be identifiable by RFLP. This is often the case, and so use of RFLP is significantly limited.

Another method to detect an allele involves the use of an oligonucleotide probe that binds specifically to sequences found in one allele, but not to other alleles. Binding of the probe to a target allele may be detected by the use of tags such as fluorescent compounds or radioisotopes. A problem of oligonucleotide probe-based methods is that to definitively ascribe a genotype it may be necessary to use a very large number of probes. Since the biophysics of polynucleotide hybridization dictate that probe length is limited (typically no more than about 65 nucleotides), where the subject gene is longer than the maximum probe length a series of different probes must be designed to cover the entire length of the gene. The number of different probes escalates greatly where the subject gene has a large number of alleles, a large number of SNPs, where the density of SNPs is high, or a combination of any of these factors.

An example of a problem in the art is the human leukocyte antigen HLA-DRB locus that is often analysed in tissue typing for organ transplantation. The locus currently has 483 identified alleles, and there are 270 nucleotides in the variable 2nd exon. Simple multiplication produces 130,410 different nucleotide sequence variations for probes that would be required to resolve a genotype at this locus. Generating such a large number of different oligonucleotide probes, and then assessing the ability of each probe to hybridise to a test sample, is clearly a significant burden. Furthermore, previously unrecognised alleles continue to be discovered thereby exacerbating the problem of providing a probe set capable of resolving an individual's HLA type.

The problems inherent in using large numbers of probes has been partially overcome by advances in solid-phase technologies that allow binding of many thousands of probes to “chips” to form a “microarray”. However, microarray technology still requires the use of many probes to identify all alleles of a gene and simply provides a more convenient format for handling large probe sets. Current probes for SNP detection are directed to physically separate regions of the target DNA molecule, and often selected where the sequence flanking the SNP is monomorphic. Use of probes such as this is known in the art as “resequencing”.

Resequencing relies on the use of specifically designed probes capable of identifying all possible SNPs. Guo et al (2002, Genome Research 12:447-457) address the problem of providing probes for HLA-typing by making 20-mer probes, with each probe designed to represent particular combinations of SNPs, rather than a single SNP. A problem with this approach is that it is not systematic, and it is necessary for a human to judiciously design the probes. Given the real possibility of error in this process it remains an uncertainty whether the probe set will identify all alleles at the end of the probe design process.

A further problem with the method provided by Guo et al is that it is necessary to include SNP sites over the length of the probe. Consideration of Table 1 of Guo et al shows that polymorphic sites are present from the 5′ end to the 3′ end of the 20-mer probes. It is known in the art that the accuracy of hybridization diminishes toward the flanks of a probe, and so it would be expected that there will be inaccuracies in the hybridization reactions using the method of Guo et al. Of particular note the probe set designed by Guo et al resulted in 32 false positive reactions among 100 hybridizations.

Accordingly, it is an aspect of the present invention to overcome or alleviate a problem of the prior art by providing a systematic method for designing probe sets capable of robustly identifying all known polymorphisms in a nucleotide sequence.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a method for identifying a set of target nucleotide sequences capable of identifying a member of a group of related nucleotide sequences, the method comprising the step of dividing the nucleotide sequence of each member of the group into a plurality of subsequences, wherein at least two of the subsequences overlap. Applicants have found that it is possible to identify a set of target nucleotide sequences useful as targets for hybridization with oligonucleotide probes by dividing the sequences under consideration into overlapping subsequences. Preferably, at least one of the subsequences overlaps with more than one other subsequence. More preferably, at least one of the subsequences overlaps with more than 2, 3, 4 or 5 other subsequences.

Advantageously, the method is amenable to automation and is proposed to be useful for providing probes capable of resolving genes having a high number of alleles and/or a high density of SNPs such as those of the major histocompatability complex (MHC), the T-cell receptor, the B-cell receptor, immunoglobulins, the killer inhibitory receptor (KIR), and the like.

In one embodiment of the method, the number of probes required for the application can be significantly reduced by identifying redundant probes, and removing or not including the redundant probes in the probe set. It has not been appreciated in the art that when analyzing related sequences for the purposes of designing a set of oligonucleotide probes, a polymorphism in one member sequence is not necessarily present in another member sequence. Accordingly, it is unnecessary to provide probes covering every combination of every polymorphism, since not all combinations necessarily exist in the group of related sequences.

In another embodiment of the method, one or more of the subsequences (and any probes derived from the subsequences) does not contain one or more polymorphic sites at, or toward, the 5′ and/or 3′ ends of the one or more subsequences. In another embodiment of the method one or more of the subsequences contains one or more polymorphic sites at, or toward, the center of the one or more subsequences. The avoidance of polymorphic sites toward the flanks of the probe, and concentrating the sites to the centre of the probe overcomes the problem of probes provide by Guo et al (2002) that apparently bind inaccurately such that a large number of false positive hybridization reactions are generated.

In another aspect the present invention provides a set of probes capable of specifically hybridizing to target nucleotide sequences identified by the methods described herein. Preferably, the probes are directed tomulti-exon coverage and are capable of providing total allele assignment.

In another aspect the present invention provides a method of identifying and/or recovering a member of a group of related nucleotide sequences using a set of probes as described herein. The method will typically utilise probes immobilised on microarray chip.

In another aspect the present invention provides a computer executable program (software) capable of executing the methods described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows hypothetical application of the method of selecting a probe set. In this case, there are three related 19-mer sequences (#1, #2 and #3). Taking the first nucleotide in the exon as 1 (i.e. the 5^(th) nucleotide in), the exon has two SNPs at positions 6 and 11 (underlined). FIG. 1A shows the related sequences divided into 9-mer subsequences, with complete overlap between the subsequences. FIG. 1B shows all subsequences pooled from related sequences #1, #2 and #3. FIG. 1C shows the set of subsequences from FIG. 1B after removal of redundant subsequences. It is emphasized that this hypothetical example does not necessarily show all the advantages of the invention, but is intended to demonstrate only the operation of a preferred form the method.

FIG. 2 shows probe sequences identified by the present invention for assignment of HLA-A*0201 (exons 2 and 3). A 25-mer probe length was chosen, with maximal overlap between probes.

DETAILED DESCRIPTION OF THE INVENTION

Applicants propose a systematic method for designing probes capable of identifying the member of a group of related nucleotide sequences. Accordingly, in a first aspect the present invention provides a method for identifying a set of target nucleotide sequences capable of identifying a member of a group of related nucleotide sequences, the method comprising the step of dividing the nucleotide sequence of each member of the group into a plurality of subsequences, wherein at least two of the subsequences overlap.

Applicants have found that it is possible to identify a set of target nucleotide sequences useful for hybridization with oligonucleotide probes by dividing the sequence under consideration into overlapping subsequences. Thus, the related group of subsequences may cover a particular locus, with each member of the related group having a different nucleotide sequence. In one form of the present method, each member of the group of related sequences is divided into a number of subsequences. Within a given member sequence, the subsequences overlap each other such that a potentially large number of subsequences may be generated. This approach is clearly distinguished from methods of the prior art that are based on the use of consecutive subsequences.

Preferably, at least one of the subsequences overlaps with more than one other subsequence. More preferably, at least one of the subsequences overlaps with more than 2, 3, 4 or 5 other subsequences.

The degree of overlap used to generate the series of overlapping probe-length subsequences may be the minimum possible. An example of minimum overlap for a series of 25-mer subsequences would be where the first subsequence covers nucleotides 1 to 25, the second subsequence covers nucleotides 25 to 50, the third subsequence covers nucleotides 50 to 75, et cetera.

The overlap may be the maximum degree of overlap possible. An example for a series of 25-mer subsequences having the maximum possible overlap would be where the first subsequence covers nucleotides 1 to 25, the second subsequence covers nucleotides 2 to 26, the third subsequence covers nucleotides 3 to 27, et cetera.

The invention includes any intermediate degree of overlap between the minimum and maximum available. However, the use of substantially maximum overlap is preferred since this requires the least amount of judgement on the part of the individual designing the probe set. The higher the degree of overlap used, the greater the ability to cover more combinations of SNPs present in the related sequences.

It is not necessary for the amount of overlap to be fixed for the use of the method with any given member of the group. It is also not necessary for the length of the subsequence to be fixed. It will be possible for the skilled person to routinely investigate the effects of varying subsequence lengths and degree of overlap between the subsequences to ascertain whether any advantage is gained.

It will be understood that where a high degree of overlap is used, a very large number of subsequences will be generated. Accordingly, a very large number of probes will be included in the probe set. While microarray chips are able to carry large numbers of probes, for economic reasons at least it is desirable to limit the number of probes required for a given analysis. In one embodiment of the method, the number of probes required for the application can be significantly reduced by identifying redundant probes, and removing or not including the redundant probes in the probe set. It has not been appreciated in the art that when analyzing related sequences for the purposes of designing a set of oligonucleotide probes, a polymorphism in one member sequence is not necessarily present in another member sequence. Accordingly, it is unnecessary to provide probes covering every combination of every polymorphism, since not all combinations necessarily exist in the group of related sequences. This approach is especially useful where the related sequences are highly polymorphic, and the present state of the art predicts that a larger-than-necessary number of probes are required to identify all theoretical members of the group. Thus, in a preferred embodiment, the method includes the step of analyzing at least a portion of the subsequences for redundancy and removing at least a proportion of any subsequences identified as redundant.

Decreasing the level of redundancy may be achieved using a subtractive approach by, for example, assuming that all polymorphisms are present in all members of the group, and generating a plurality of subsequences based on that assumption. Subsequently, the plurality of subsequences is analyzed for the presence of redundant sequences, which are then removed to leave the set of unique target nucleotide sequences. It will be appreciated that the set of target nucleotide subsequences has the same capability of identifying every member of the group as the larger set of subsequences that are generated on the assumption that all polymorphisms are present in all members.

Alternatively, an additive method may be used where the plurality of probe-length sequences is incrementally generated, one by one, with each newly generated subsequence being analyzed for redundancy in light of all previously generated subsequences. If a newly generated subsequence is found to be redundant it is not added to the set of target nucleotide sequences, otherwise it is included in the set of target nucleotide sequences. Whether an additive or subtractive method is used, the end result is the same: a set of subsequences having no redundancy, or a reduced level of redundancy, is generated that is capable of identifying all members of the group of related sequences.

It is desirable to limit the number of probes required to identify a member sequence for a number of reasons. The cost of synthesizing probes and producing microarray chips to carry those probes is a significant consideration in the economic viability of implementing a method for identifying a nucleotide sequence. This is the case whether it is for purely research purposes, or for a high throughput commercial application such as in a pathology laboratory. Particularly, where a nucleotide sequence can have many alternative forms (i.e. where the number of members in the group of related sequences is high), the prior art methods require a commensurately high number of different specific probes. Thus, to screen for the presence of a single member nucleotide sequence it may be necessary to use hundreds, or even thousands of individual probes depending on the length of the sequence to be interrogated.

Another reason for limiting the number of probes necessary for identifying a member nucleotide sequence relates to the practical limits of certain probe hybridization methods. For example, a standard dot blot apparatus may have only 64 wells for sample application, thereby restricting the user to only 64 different probes, and therefore the ability to identify only 64 different nucleotide sequences per run. A further example is where a microarray system is used to identify a very large number of alternative forms of a nucleotide sequence. At present, a standard microarray chip can hold up to 500,000 different oligonucleotide probes. While this may appear to be ample, for some applications this number is insufficient and it would be necessary to prepare multiple chips to accommodate all probes.

In one embodiment of the method, one or more of the subsequences (and any probes derived from the subsequences) does not contain one or more polymorphic sites at, or toward, the 5′ and/or 3′ ends of the one or more subsequences. In another embodiment of the method one or more of the subsequences contains one or more polymorphic sites at, or toward, the center of the one or more subsequences. The avoidance of polymorphic sites toward the flanks of the probe, and concentrating the sites to the centre of the probe overcomes the problem of probes provide by Guo et al (2002) that apparently bind inaccurately such that a large number of false positive hybridization reactions are generated.

The related nucleic acid sequences can be genomic, RNA, cDNA, or cRNA. Genomic DNA samples are usually subject to amplification before application to an array using primers flanking the region of interest. Genomic DNA can be obtained from virtually any tissue source (other than pure red blood cells). For example, convenient tissue samples include-whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. Amplification of genomic DNA containing a polymorphic site generates a single species of target nucleic acid if the individual from the sample was obtained is homozygous at the polymorphic site or two species of target molecules if the individual is heterozygous.

The DNA may be prepared for analysis by any suitable method known to the skilled artisan, including by PCR using appropriate primers. Where it is desired to analyze the entire genome, the method of whole genome amplification (WGA) may be used. Commercial kits are readily available for this method including the GenoPlex® Complete WGA kit manufactured by Sigma-Aldrich Corp (St Louis, Mo., USA). This kit is based upon random fragmentation of the genome into a series templates. The resulting shorter DNA strands generate a library of DNA fragments with defined 3 primed and 5 primed termini. The library is replicated using a linear, isothermal amplification in the initial stages, followed by a limited round of geometric (PCR) amplifications. WGA methods are suitable for use with purified genomic DNA from a variety of sources including blood cards, whole blood, buccal swabs, soil, plant, and formalin-fixed paraffin-embedded tissues.

mRNA samples are also often subject to amplification. In this case amplification is typically preceded by reverse transcription. Amplification of all expressed mRNA can be performed as described in WO 96/14839 and WO 97/01603. Amplification of an RNA sample from a diploid sample can generate two species of target molecule if the individual from whom the sample was obtained is heterozygous at a polymorphic site occurring within expressed mRNA.

As will be apparent, the nucleotide subsequences identified by the method may be subsequently used to design a probe set capable of identifying all currently identified members of the group of related sequences. As used herein the term “target nucleotide sequence” means a sequence against which a substantially specific probe may be generated. The generation of probes is discussed further infra, however the probe is typically an oligonucleotide probe capable of hybridizing to the target nucleotide sequence.

Applicants have found that even where the group of related sequences has a large number of members, and/or where the members have a large number of polymorphic bases, and/or where the polymorphic bases have more than two alternative forms, it is possible to produce a probe set capable of definitively identifying any member of the group using a number of probes significantly less than that previously considered in the art to be necessary. The method may be used, for example, to produce a probe set capable of identifying any given allele of a gene locus, and is especially useful where the number of alleles is very high. By contrast, Guo et al (2002) do not disclose a practical and robust method for designing probes for multi-exon coverage capable of providing total allele assignment.

The skilled person will understand that the length of the probe-length subsequences may be any length that provides the ability to discriminate between the members of the group of related sequences.

Probes used for microarray applications are typically about 25 nucleotides in length, however longer and shorter probes are contemplated to be useful in the context of the invention. A lower useful length may be determined by the need for sufficient nucleotides to provide specificity of binding, and may be from about 10 nucleotides to about 15 nucleotides. Probes of a less than 15 nucleotides could be contemplated where a “sub-genome” is under test. An example of this is where single haploid chromosomes are under test, and sequence detection specificity does not require a probe length needed to analyze the approximately 3 billion nucleotides in the entire genome of a human. The upper limit may be determined by physical constraints relating to the need to melt double-stranded regions and anneal single strands of polynucleotide. This may be from about 30 to about 50 nucleotides. The upper limit may vary according to the proportion of C/G bases given the higher melting temperatures needed to separate these bases in a duplex, as compared with an A/T pairing. While there may be practical upper and lower limits for the length of probe, these limits will vary according to the specifics of the application and the skilled person will be able to identify the probe of most appropriate length by routine empirical experimentation.

It will be understood that the method may be applied to any situation where it is necessary to discriminate between a number of related nucleotide sequences. As used herein, the term “nucleotide sequence” and variations thereof is intended to include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sequences. The related nucleotide sequences may be any group of nucleotide sequences that exhibit a minimum level of sequence identity. Preferably the sequences have an identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99%. The identity may be even higher than 99% where, for example, the related sequences are long, and there are a series of SNPs scattered throughout.

The related sequences may be protein coding, non-protein coding, or a combination of protein coding and non-protein coding.

The related sequences may be derived from diploid, haploid, triploid or polyploid material, or provide information on the diploid, haploid, triploid or polyploid state.

Where information is sought on the haploid state, the present methods are useful for providing probes that can provide definitive DNA allele assignment to haplotype stratification. The concept of locus allelism is known in the art, however it has not previously been appreciated that allelism of loci that bound regions, including alleles that involve synonymous changes, are contributory to haplotype stratification. Thus, probes for genomic (diploid) DNA can inform about haplotypic (cis phase) multi-allele assignment. Specifically, synonymous alleles are a unit in multilocus chromosomal haplotypic segment. Probes generated by the methods described herein that characterise locus allelism contribute to revelation of patterns of multilocus co-allelism, which is haplotypy. This concept is exemplified by telomeric G and F loci. There are 23 alleles at HLA-G and 20 at HLA-F. These 43, combined with the 120 at centromeric DPB1 locus, as well as those many in between will assist in assigning the finite multi-locus allelic variations as haplotypes spanning the <4 Mb MHC region.

The related sequences may be natural or synthetic. They may be from any organism including an animal, plant, microorganism, bacterium, or virus.

In one form of the invention, the related sequences are directed to the same region of the genome. For example, the region from the first nucleotide of an exon to the last nucleotide of the exon. In this case, and where a 25-mer probe is to be used, the probe may be designed such that the 13^(th) nucleotide of the probe (i.e. the central nucleotide) is directed to the first nucleotide of the exon. Thus, where the first nucleotide is G, the 13^(th) nucleotide of the probe will be C. It will be apparent that the flanking 12-mer regions of the probe will be directed in one case to the pre-exon region and in the other case, further into the exon.

The general operation of one embodiment of the method can be demonstrated by consideration of the greatly simplified example shown in FIG. 1. This demonstration is directed to 3 related nucleotide sequences (#1, #2 and #3), with the exon starting at the 5^(th) nucleotide in from the left hand or 5′ end (i.e. A). Taking the first nucleotide in the exon as 1, the exon has two SNPs at positions 6 and 11 (underlined). Subsequences of 9 nucleotides were used, with there being complete overlap in the subsequences. Thus, the first subsequence commences at position −4 and terminates at position +5.

As will be apparent from FIG. 1A, each related sequence is divided into 11, 9-mer subsequences. This provides a total of 33 subsequences (FIG. 1B). Duplicate subsequences are removed to leave 17 unique subsequences (FIG. 1C). The skilled person will understand that the probe sequences do not need to be complimentary if the original target molecule was a double-stranded molecule. In that case, the nucleotide sequence can be directly used as the probe sequence or complimented to ACAGGGGTGTCGTGCAAAGMCCTC, depending on the target generation strategy chosen by the skilled artisan. Thus, the probe can be directed to either strand, or both, on the array if dsDNA is used in final target generation).

It should be appreciated that this example is provided simply to demonstrate the steps required to generate a probe set capable of distinguishing the members of a group of related nucleotide sequences according to one form of the present invention. In this case, a reduction in probe number of about 50% is achieved. In more complex systems, the reduction in probe number will be significantly greater, possibly in excess of 95%.

The methods of the present invention will allow analysis of many variations in nucleotide sequences including deletions, substitutions, additions and the like. In one form of the invention the related nucleotide sequences are identical except for the presence of SNPs.

While the SNPs may be present at any density, the methods provide greater advantages where the SNPs are present at a high density. Preferably the density is such that two or more SNPs are present within a probe length region of the nucleotide sequence. The ability to distinguish related nucleotide sequences that include SNPs at high density has previously been problematic since it has hitherto been thought necessary to provide a large number of probes to cover every combination of SNPs in a given region. This has especially been an issue in designing probe sets for HLA typing where 20% to 50% of the nucleotides in HLA exons are polymorphic, and often the polymorphic sites are clustered. This has resulted in the prior art predicting that a practically infeasible number of different probes would be required to definitively ascribe an HLA type to an individual.

It will be clear that while the number of related nucleotide sequences in the group may be as low as two, the method provides an increased advantage where the number of related nucleotide sequences is high. In a preferred form of the method the number of related nucleotide sequences in the group of related nucleotide sequences is more than 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000. The present invention is particularly applicable where the number of related nucleotide sequences is high and the density of SNPs is high.

In a preferred form of the method, the related nucleotide sequences are alleles of a gene. It is known that a human gene encoding the same protein may have different sequences (alleles) in different individuals. The proportion of the gene analyzed can be any proportion capable of providing allele-specific information. For example, polymorphic sites are often distributed non-randomly across the length of exons. Thus it may be necessary to direct probes only to certain discrete regions of a gene.

While most genes have only several alleles, some genes have a very high number. Examples of genes having high numbers of alleles are mainly those involved in the immune system, where hypervariability is a common feature. Exemplary genes include those of the major histocompatability complex (MHC), the T-cell receptor, the B-cell receptor, immunoglobulins, the killer inhibitory receptor (KIR), and the like. It will be understood however, that the methods described herein will be useful for any group of related nucleotide sequences, but that a greater advantage is gained where the related nucleotide sequences are hypervariable. A greater advantage still is provided where the hypervariability exits as high density SNPs.

As mentioned supra, MHC genes are extremely polymorphic. Class I and II MHC transmembrane proteins make up the Human Leukocyte Antigen (HLA) system that is used in tissue typing for the purposes of assessing transplant compatibility. Class I proteins are encoded by three loci: HLA-A, HLA-B and HLA-C that currently recognize 309, 563 and 167 alleles respectively.

Class II proteins have an alpha and beta chain, and are encoded by the loci DR, DQ and DP. The DR loci comprise 3 alleles for alpha and 483 for the beta chain. The DQ loci comprise 25 alleles for alpha and 56 for beta. The DP loci comprise 20 alleles for alpha and 107 for beta. It will therefore be noted that for the Class I region alone, there are many combinations of alleles that provide the HLA type of an individual.

Historically, HLA-based tissue typing was performed serologically using antibodies specific for those HLA antigens that have been identified in the human population. Most HLA typing is now performed by DNA methods, for high level allele assignment by sequencing, or sequence-equivalent methods. Such DNA typing, promises to improve the sensitivity and specificity of tissue typing. However, a problem with attempting to identify all HLA alleles by DNA-based methods (involving oligonucleotide sequences as probes) is that a very large number of probes is required to cover all possible alleles. The present invention alleviates this problem by providing probe sets that are manageable in number, while still capable of identifying all known alleles.

While the HLA-DR beta loci is currently recognized to comprise 483 alleles, it may appear that only 483 probes are necessary (one for each allele) until it is understood that each allele is a unique combination/pattern of SNPs distributed across all exonic nucleotides. The art has generally considered that the presence of even di-allelic SNPs is a significant problem in probe design given that current microarray SNP detection practice in which where a 25-mer oligonucleotide probe is used, the 12-mers flank the 13th position SNP allele. Therefore, where the flanking region(s) are non-monomorphic the art has hitherto thought it necessary to include probes that cover every SNP in every known combination within the 25-mer region even though not all exist in nature. It is accepted in the art that any polymorphic site requires 4 to the power of the number of alleles known to occur at that site. Thus, if the flanking 12-mers encompass two SNPs each, in both flanks, then the number of probes required to type the 13th position SNP is at least 4 to the power of 2=16.

Applicant's approach is divergent and is based on the recognition that not all sites that are polymorphic in any probe-length subsequence is present in all alleles of a HLA locus.

Without wishing to be limited by theory in any way, it is proposed that for HLA loci the theoretical possibilities are some 5-20 fold greater than the observed allelic sequences. An example of complex high SNP density loci are the HLA-DRB region loci (Expressed DRB1, DRB3, DRB4, DRB5; pseudogenes-not expressed DRB2, DRB6, DRB7, DRB8, DRB9). There are (some) 483 identified alleles among both categories of genes in this region. There are 270 nucleotides in the variable 2nd exon. Simple multiplication produces 130,410 different probes that would be required to resolve a genotype at this locus. There may be two main reasons for this observation: (i) combinations of SNPs exhibit linkage disequilibrium because they are inherited on chromosomal lengths that ensures non-randomness of SNP association; and (ii) populations have experienced ‘bottleneck’, resulting in the disappearance of some multi-SNP alleles, and the relative increase in frequency of others, influenced by population genetic factors such as natural selection, propensity for recombination, et cetera.

The present invention makes it possible to reduce the number of probes necessary for the identification of a genotype in a highly polymorphic system (such as HLA loci) such that all probes required to identify every allele may be immobilized on a single typical microarray chip.

It will be understood that the final number of probes required to definitively identify an allele will depend on the locus under consideration. However, in a preferred form of the method it is expected that more than a 50%, 60%, 70%, 80%, 90% or 95% reduction in probe number may be possible relative to the theoretical number of probes thought to be necessary.

While it is contemplated that maximum advantage in terms of minimising probe number will be gained where all redundant subsequences are removed, it is not essential to the invention that all are removed. Indeed, in some instances it is advantageous for some redundancy in subsequences to be maintained, in that an internal quality control mechanism results. Redundancy in the probe set can result from the fact that redundancy occurs across loci. Redundant probes relating to redundancy across loci may therefore be maintained in a probe set provided by the present invention for the purposes of quality control. As an example, where a probe list is generated for the assignment of allele types at HLA Class I and Class II loci and of genes and allele types at the KIR loci, about 34,500 probes are identified. The list identifies variations involving hypervariable exons 2 and 3 at HLA Class I loci (A, B, C) and exon 2 at Class II loci (DRB, DQB, DPB), and all known variations at up to 10 exons at KIR loci. In the list of probes, there are 2167 duplicated sequences due to direct repeats of sequences present when comparing HLA-A, -B, and -C, or DPB, DQB, and DRB, e.g.

!Probe Tag? Probe Sequence 5522A_E3_232_2_25 TCCGCAGATACCTGGAGAACAGGAA 15458C_E3_232_4_25 TCCGCAGATACCTGGAGAACAGGAA 9492B_E3_13_17_25 TCCAGAGGATGTTTGGCTGCGACCT 13765C_E3_13_10_25 TCCAGAGGATGTTTGGCTGCGACCT 22138R_E2_155_21_25 TGTCGCCGAGTACTGGAACAGCCAG 17957Q_E2_155_9_25 TGTCGCCGAGTACTGGAACAGCCAG 21088R_E2_105_3_25 TTCGACAGCGACGTGGGGGAGTTCC 17442Q_E2_105_3_25 TTCGACAGCGACGTGGGGGAGTTCC 16011P_E2_99_1_25 TTCGACAGCGACGTGGGGGAGTTCC

Where probes are labelled in the following manner

a=consecutive probe number F=either A, B, C, P, Q, R, K E=exon c=exon number d=first base of 25-mer in exon e=1-30, 1 is the reference (consensus), unique allele types follow consecutively f=probe length.

The replicate probe sequences are retained in one form of the invention to contribute to both technical and genetic components of quality assurance. Specifically, where there is a bona fide hybridisation with one probe consistent with reactivity to all other probes identifying an allele at the first locus, but in which the same probe sequence is not an integral component of either allele at a second locus, then there will be reactivity in the replicate distinct from those reflecting the alleles at the second locus.

As an example of the operation of this internal quality control mechanism, the lowest level of resolution is the allele lineage, or family. Considering DRB there are 13 lineages (*01, *03, *04, *07, *08, *09, *10, *11, *12, *13, *14, *15, *16). By including probes for all four DRB expressed loci, the presence or absence of DRB3, DRB4 and DRB5 provides information on the lineage type of DRB1 alleles, independent of DRB1 probe reactivity.

In the context of the present invention, the term “redundant” is intended to mean that if the sequence is removed from the first set of subsequences there is no appreciable difference in the ability to identify a member of the group of related nucleotide sequences. Redundancy may be considered as complete (i.e. two subsequences are identical in nucleotide sequence) or incomplete (e.g. the two subsequences are physically non-identical, but are functionally identical). Thus, depending on the hybridisation conditions used, two different probes may bind to a single nucleotide sequence and are therefore functionally identical. This would be expected where hybridisation conditions are of a relatively low stringency.

The non-redundant or reduced redundancy sequences are generated based on the alleles previously identified using DNA sequencing. If a new allele is identified that contains a new polymorphism, then additional target sequences may need to be included in the probe set to ensure detection of that new polymorphism. If the new polymorphism occurs in a target sequence that was previously found to be redundant, then in light of the knowledge of the new polymorphism, that target sequence becomes necessary as a probe target and therefore non-redundant.

In one form of the method, the method is amenable to automation. Methods of the prior art such as Guo et al (2002) design probes based on the careful consideration of all related nucleotide sequences in an effort to identify probes that cover all observed combinations of SNPs. This is of course very labour intensive, and the success or failure dependant on the expertise of the individual performing the analysis. The task of designing probes may become practically infeasible if the number of related sequences is very large, or the number of alleles is very large. By contrast, the present methods are particularly amenable for implementation on a computer in the form of software-based probe set design.

The method may include a combination of different subsequence lengths and different levels of overlap between the subsequences. In a highly preferred form of the invention the subsequence is about 25 nucleotides in length, and the degree of overlap is maximal.

The related sequences may include sequences from all known alleles of a gene. Alternatively, the related sequences may include known and hitherto unknown sequences. For example, it may be known that a polymorphism is found at a given position in a gene, and that the position can have one of two alternative forms (e.g. A or T). It will be possible to include “hypothetical” sequences where a G or C is present in that position. Alternatively, where a given position is not known to have any polymorphisms but is suspected to, probes directed to the three alternative forms may be included in the probe set. Furthermore, the invention will allow the detection of new combinations of SNPs that result in a new allele. These approaches are very probe-demanding, and use of the present invention makes it practically feasible given the vast reduction in probe numbers required. The chance for finding new alleles will be greater where maximum overlap between the subsequences is used.

It will be appreciated that the presence of a hitherto unrecognised allele may also be discovered by the internal quality control mechanisms as discussed supra. Probe reactivity discordance with known alleles will signal the presence of either an error in assay, or the presence of a new allele.

As discussed supra, the allele analysed may be directed to protein-coding regions exclusively, or noncoding regions exclusively. Alternatively, a combination of noncoding and protein-coding regions may be used.

In another aspect the present invention provides a set of probes capable of specifically hybridizing to target nucleotide sequences identified by the methods described herein. In one form of the invention, the probe set has a lower level of redundancy than a probe set designed by methods known in the art.

Given the target subsequences, the skilled person will be capable of synthesizing probes capable of hybridising with each target subsequence. The probes are substantially complimentary to the non-redundant sequences identified. The probes may be sense or antisense if the target is generated from a double stranded template. The probes can be made by any method known to the skilled artisan, although the final use of the probes will likely dictate the most appropriate method. For example where the probes are for use in a microarray environment, they may be synthesized in situ on the glass or nylon wafer forming the array solid support matrix. For other applications, the probes may be synthesized on an automated apparatus such as the Beckman 1000M DNA synthesizer and subsequently used for methods such as PCR to detect an allele. Alternatively, the probe may be coupled to a solid support after manufacture.

It is well within the ability of the skilled person to investigate whether any advantage is gained by the use of modified nucleotides in probes designed by the instant methods, such as locked nucleic acids.

For the purposes of quality assurance, the probe set optionally includes a paired “mismatch” probe for each probe on the array that perfectly matches its target sequence. The mismatch probe contains a single mismatch located directly in the middle of the 25-base probe sequence. While the perfect match probe provides measurable fluorescence when sample binds to it, the paired mismatch probe is used to detect and eliminate any false or contaminating fluorescence within that measurement. The mismatch probe serves as an internal control for its perfect match partner because it hybridizes to non-specific sequences about as effectively as its counterpart, allowing spurious signals, from cross hybridization for example, to be efficiently quantified and subtracted from a gene expression measurement or genotype call.

The probe may include a label to facilitate detection. Exemplary labels include Cy5, Cy3, FITC, rhodamine, biotin, DIG and various radioisotopes.

A probe sequence list generated according to the present invention can be expanded to include additional allelic variation at other exons within the mRNA transcript, at sequences intervening or flanking the exons, including introns, 5′ and 3′ untranslated regions, and intergenic regions.

In another aspect the present invention provides a method of identifying a member of a group of related nucleotide sequences using a set of probes as described herein. One way of achieving this is using microarray technology. Thus, another aspect the invention provides a set of probes as described herein immobilized on a solid matrix. An exemplary embodiment of this form of the invention is found in the GeneChip® technology marketed by Affymetrix®. This technology relies on a photolithographic process by coating a 5″×5″ quartz wafer with a light-sensitive chemical compound that prevents coupling between the wafer and the first nucleotide of the DNA probe being created. Lithographic masks are used to either block or transmit light onto specific locations of the wafer surface. The surface is then flooded with a solution containing either adenine, thymine, cytosine, or guanine, and coupling occurs only in those regions on the glass that have been deprotected through illumination. The coupled nucleotide also bears a light-sensitive protecting group, so the cycle can be repeated. Other methods of immobilizing probes are provided by a number of companies including Oxford Gene Technology (Oxford, U.K.), Agilent Technologies (Palo Alto, Calif., U.S.A.) and Nimblegen Systems Inc (Madison, Wis., U.S.A).

The probes of the present invention are useful not only for identifying a member of a group of related nucleotide sequences, but also for the recovery of the member so identified. Accordingly, one form of the method further comprises the step of recovering a member of a group of related nucleotide sequences using a probe set as described herein. In the context of the present invention, the term “recover” includes the physical separation of the member identified by (or bound to) a probe forming part of a probe set of the present invention from at least one other member of a group of related nucleotide sequences. Advantageously, the recovered member can be analysed to provide genotypic and/or phenotypic information on the subject from which the member is derived.

The method may comprise the steps of exposing the probe to the group of related nucleotide sequences under conditions allowing a probe of the probe set to bind to a nucleotide sequence of the group of related nucleotide sequences to form a probe/nucleotide sequence complex, and substantially isolating the probe/nucleotide sequence complex.

The skilled person is familiar with identifying conditions allowing binding of a nucleic acid probe to a target nucleotide sequence. It is also within the capabilities of the skilled person to identify conditions conducive to the specific binding of a nucleic acid probe to a target nucleotide sequence. Physical parameters of the reaction solution such as temperature, ionic strength and pH may be manipulated such that binding takes place on a specific or non-specific basis.

The skilled person is also aware of many methods for the substantial isolation of a probe/nucleotide sequence complex. Recovery of molecules using reagents that are chemically reciprocal to the target, such as nucleotide sequence by anti-sense sequence, or vice versa; are well known across many chemistries. Typically, a probe is attached to a solid phase such as a chromatographic matrix, a bead (for example, a magnetic bead), or a planar glass surfaces (such as those used microarray formats, for example SuperEpoxy, SuperAmine, SuperAldehyde and SuperNitro manufactured by Telechem International Inc). The attached probe is then exposed to a solution containing a mixture of nucleic acid sequence fragments, and binding of the probe to nucleic sequence allowed to occur. The probe/nucleic acid sequence complex is then separated from unbound molecules by a suitable method. For example, where the probe is bound to a magnetic bead, the magnetic beads (with at least some having bound nucleic acid sequence fragment) are separated by the application of a magnetic field to the reaction solution.

It will be understood that in some situations the probe/nucleotide sequence complex can be recovered without attachment of either reactant to a solid phase. For instance, probe/nucleotide sequence complexes may be separated in the fluid phase of electrophoresis. A DNA fragment bound to a probe will migrate at a different rate to a fragment of the same, or similar, electrophoretic mobility.

Once the probe/nucleotide sequence complex is substantially isolated, the nucleotide sequence may be eluted from the probe. Typically, it is the elution step that is manipulated to increase or decrease the specificity of the probe/nucleotide sequence binding reaction. Elution may be achieved by altering any one of more of the following parameters: temperature, ionic strength and pH. Elution may also be controlled with the use of detergents or other additives.

The recovered nucleotide sequence may be analyzed by any appropriate method to obtain any required information. The analysis may include any one or more of the following characteristics: nucleotide sequence, AT content, CG content, length, secondary structure, ability to bind to a protein, ability to bind to another nucleic acid sequence, ability to be cleaved by an endonuclease, methylation status, and the like. Typically, however, the analysis will be nucleotide sequence analysis.

The recovered nucleic acid sequences may be any length, but in some forms of the invention at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 bases long. In other forms of the invention the recovered nucleic acid sequence is at least 100, 200, 300, 400, or 500 bases long.

The recovered nucleic acid sequence may be used for any reason, however it is typically used for providing genotypic and/or phenotypic information on a subject. The probe sets provided by the present invention are, in some embodiments, capable of binding to every known allele of a given gene. For example, if it is desired to read the nucleotide sequence of a certain fragment of genomic DNA, and that fragment of genomic DNA included a number of sites at which mutations were possible, then that fragment may be recovered from any subject irrespective of the presence or absence of any mutation(s). As discussed supra a particular advantage is gained for the recovery of fragments having a high density of SNPs, such as fragments of HLA-MHC genes, or KIR genes.

In one embodiment, the method is used for the isolation of exomic nucleic acid sequences from a subject. As is now understood, the proportion of genomic DNA that actually codes for protein is small, and the present invention may be used to extract just that exomic proportion from the whole of a subject's genomic DNA for subsequent analysis. This approach requires significantly less sequence analysis than would be otherwise required where the whole genome is sequenced.

In another aspect the present invention provides a computer executable program (software) capable of executing the methods described herein. While the present invention may be implemented manually, it is preferably performed on a personal computer under the instruction of appropriate software. Given the disclosure herein, the skilled person will be enabled to write appropriate code to execute the method. Example pseudo-code for the 0101 allele DRB1 locus follows:

[AWAIT USER INPUT] (IF) Mouse_Click Event detected on the Grid interface; [DETERMINE]grid row and grid column of the Click; /* Since all sequences are displayed in tabular format, they are also stored in tabular format as a memory object according to the following: ReferenceNameArray[position 0] = “DRB10101”; ReferenceBasisArray[position 0] = “TGTCCCCA....”; which in memory forms a tabular structure like this: ReferenceNameArray ReferenceBasisArray Index 0: “DRB01*010101” “ TGTCCCCA....” Index 1: “DRB01*010102” “ TGTCCCCC....” Index 2: “DRB01*010103” “ TGTCCCCC....” */ [DETERMINE] ReferenceBasisArray base range (25 mers) using grid column click value as index. [DETERMINE] ReferenceNameArray using grid row click as index /** how to determine the range of 25? If the ReferenceBasisArray (ie: array of all bases) contains 150 bases, then use the grid column click value to determine the middle point. ie:

hence if the user clicks on column 12, then our range becomes, min = middle_point - 12, max = middle_point + 12; **/ [EXTRACT] 25 bases from each ReferenceBasisArray Record (IF) base is different to Reference Record [HIDE/DISCARD] row else [DISPLAY ROW] ------------------------------------------------------

The software may have the facility to investigate the effects of a range of parameters on the number of probes required to resolve a specific allele. In this way, it may be possible to further decrease the number of probes required. For example, the software may allow the user to define the length of the probe-length subsequence, the degree of overlap of the subsequences, the rules for defining whether two subsequences are redundant and the like. Indeed, the software may include algorithms to automatically trial a range for each parameter to give the lowest number of probe-length subsequences (and therefore the number of probes in the probe set). A probe may also be removed from the probe set if it is considered likely to have significant secondary structure, or too high or too low a melting temperature such that it will not reliably hybridise to the relevant target. A probe may be removed from the probe set on the basis of empirical probe optimisation experiments demonstrating a lack of suitability.

It will be appreciated that the present invention will have application in a wide range of technical fields. It is anticipated that the field of medicine will gain particular advantage, where the method may be used for genotyping individuals. The methods will be particularly useful in transplantation tissue typing (e.g. using the HLA genes, KIR genes, minor Histocompatability loci, and the like), as well as pharmacogenomics, DNA “fingerprinting” and the like. The probes may be used for any application comprising in situ hybridisation, slot blot, dot blot, colony hybridization, plaque hybridization, Northern blotting, Southern blotting, as well as microarray applications,

It is anticipated that the invention will be useful in any application where it is necessary or desirable to reduce the number of unique probes required for analysis of a nucleotide sequence, and not only in the area of microarray analysis. The invention will be applicable even where the numbers of probes required to undertake a task in identifying a particular nucleotide sequence amongst a number of others are not so great as to extend beyond the capacity of a chip. Minimisation of probe numbers will allow tests for other loci to be included on the one chip such that an increase number of loci can be tested for on the one chip. It is of course less costly to run one chip as compared with 20.

It is anticipated that applications will extend to use in non-human animals such as primates, for example in the pre-clinical pharmacogenomic assessments of candidate pharmaceuticals. The invention is also contemplated to be useful for testing of animals having economic importance (such as cattle, poultry and the like), for example in breeding programs to improve parameters such as lean muscle content.

The present invention will now be further described by reference to the following non-limiting example. The skilled person will understand that the HLA loci are some of the most variable loci found in nature. It will be appreciated that a method able to be operable for an HLA locus, then any other locus will be operable.

EXAMPLE 1 Identification of Oligonucleotide Probe Set for Definitive Genotyping of the HLA-DRB Locus Outline of Protocol

The DRB locus of HLA was analyzed by the present methods to identify a probe set capable of identifying any known allele of the locus. The DRB locus has the following known alleles:

DRB1*010101, DRB1*010102, DRB1*010103, DRB1*010201, DRB1*010202, DRB1*010203, DRB1*010204, DRB1*0103, DRB1*0104, DRB1*0105, DRB1*0106, DRB1*0107, DRB1*0108, DRB1*0109, DRB1*0110, DRB1*0111, DRB1*0112, DRB1*0113, DRB1*030101, DRB1*030102, DRB1*030201, DRB1*030202, DRB1*0303, DRB1*0304, DRB1*030501, DRB1*030502, DRB1*0306, DRB1*0307, DRB1*0308, DRB1*0309, DRB1*0310, DRB1*0311, DRB1*0312, DRB1*0313, DRB1*0314, DRB1*0315, DRB1*0316, DRB1*0317, DRB1*0318, DRB1*0319, DRB1*0320, DRB1*0321, DRB1*0322, DRB1*0323, DRB1*0324, DRB1*0325, DRB1*0326, DRB1*0327, DRB1*0328, DRB1*040101, DRB1*040102, DRB1*0402, DRB1*040301, DRB1*040302, DRB1*0404, DRB1*040501, DRB1*040502, DRB1*040503, DRB1*040504, DRB1*0406, DRB1*040701, DRB1*040702, DRB1*040703, DRB1*0408, DRB1*0409, DRB1*0410, DRB1*0411, DRB1*0412, DRB1*0413, DRB1*0414, DRB1*0415, DRB1*0416, DRB1*0417, DRB1*0418, DRB1*0419, DRB1*0420, DRB1*0421, DRB1*0422, DRB1*0423, DRB1*0424, DRB1*0425, DRB1*0426, DRB1*0427, DRB1*0428, DRB1*0429, DRB1*0430, DRB1*0431, DRB1*0432, DRB1*0433, DRB1*0434, DRB1*0435, DRB1*0436, DRB1*0437, DRB1*0438, DRB1*0439, DRB1*0440, DRB1*0441, DRB1*0442, DRB1*0443, DRB1*0444, DRB1*0445, DRB1*0446, DRB1*0447, DRB1*0448, DRB1*0449, DRB1*0450, DRB1*0451, DRB1*0452, DRB1*070101, DRB1*070102, DRB1*0703, DRB1*0704, DRB1*0705, DRB1*0706, DRB1*0707, DRB1*0708, DRB1*0709, DRB1*080101, DRB1*080102, DRB1*080201, DRB1*080202, DRB1*080203, DRB1*080302, DRB1*080401, DRB1*080402, DRB1*080403, DRB1*080404, DRB1*0805, DRB1*0806, DRB1*0807, DRB1*0808, DRB1*0809, DRB1*0810, DRB1*0811, DRB1*0812, DRB1*0813, DRB1*0814, DRB1*0815, DRB1*0816, DRB1*0817, DRB1*0818, DRB1*0819, DRB1*0820, DRB1*0821, DRB1*0822, DRB1*0823, DRB1*0824, DRB1*0825, DRB1*0826, DRB1*0827, DRB1*0828, DRB1*0829, DRB1*090102, DRB1*0902, DRB1*0903, DRB1*0904, DRB1*100101, DRB1*100102, DRB1*110101, DRB1*110102, DRB1*110103, DRB1*110104, DRB1*110105, DRB1*1102, DRB1*1103, DRB1*110401, DRB1*110402, DRB1*1105, DRB1*110601, DRB1*110602, DRB1*1107, DRB1*110801, DRB1*110802, DRB1*1109, DRB1*1110, DRB1*1111, DRB1*111201, DRB1*111202, DRB1*1113, DRB1*1114, DRB1*1115, DRB1*1116, DRB1*1117, DRB1*1118, DRB1*111901, DRB1*111902, DRB1*1120, DRB1*1121, DRB1*1122, DRB1*1123, DRB1*1124, DRB1*1125, DRB1*1126, DRB1*112701, DRB1*112702, DRB1*1128, DRB1*1129, DRB1*1130, DRB1*1131, DRB1*1132, DRB1*1133, DRB1*1134, DRB1*1135, DRB1*1136, DRB1*1137, DRB1*1138, DRB1*1139, DRB1*1140, DRB1*1141, DRB1*1142, DRB1*1143, DRB1*1144, DRB1*1145, DRB1*1146, DRB1*1147, DRB1*1148, DRB1*1149, DRB1*1150, DRB1*1151, DRB1*1152, DRB1*1153, DRB1*1154, DRB1*120101, DRB1*120102, DRB1*120201, DRB1*120202, DRB1*120302, DRB1*1204, DRB1*1205, DRB1*1206, DRB1*1207, DRB1*1208, DRB1*1209, DRB1*1210, DRB1*1211, DRB1*130101, DRB1*130102, DRB1*130103, DRB1*130201, DRB1*130202, DRB1*130301, DRB1*130302 DRB1*1304, DRB1*1305, DRB1*1306, DRB1*130701, DRB1*130702, DRB1*1308, DRB1*1309, DRB1*1310, DRB1*1311, DRB1*1312, DRB1*1313, DRB1*131401, DRB1*131402, DRB1*1315, DRB1*1316, DRB1*1317, DRB1*1318, DRB1*1319, DRB1*1320, DRB1*1321, DRB1*1322, DRB1*1323, DRB1*1324, DRB1*1325, DRB1*1326, DRB1*1327, DRB1*1328, DRB1*1329, DRB1*1330, DRB1*1331, DRB1*1332, DRB1*1333, DRB1*1334, DRB1*1335, DRB1*1336, DRB1*1337, DRB1*1338, DRB1*1339, DRB1*1340, DRB1*1341, DRB1*1342, DRB1*1343, DRB1*1344, DRB1*1345, DRB1*1346, DRB1*1347, DRB1*1348, DRB1*1349, DRB1*1350, DRB1*1351, DRB1*1352, DRB1*1353, DRB1*1354, DRB1*1355, DRB1*1356, DRB1*1357, DRB1*1358, DRB1*1359, DRB1*1360, DRB1*1361, DRB1*1362, DRB1*1363, DRB1*1364, DRB1*1365, DRB1*1366, DRB1*140101, DRB1*140102, DRB1*1402, DRB1*140301, DRB1*140302, DRB1*1404, DRB1*140501, DRB1*140502, DRB1*1406, DRB1*140701, DRB1*140702, DRB1*1408, DRB1*1409, DRB1*1410, DRB1*1411, DRB1*1412, DRB1*1413, DRB1*1414, DRB1*1415, DRB1*1416, DRB1*1417, DRB1*1418, DRB1*1419, DRB1*1420, DRB1*1421, DRB1*1422, DRB1*142301, DRB1*142302, DRB1*1424, DRB1*1425, DRB1*1426, DRB1*1427, DRB1*1428, DRB1*1429, DRB1*1430, DRB1*1431, DRB1*1432, DRB1*1433, DRB1*1434, DRB1*1435, DRB1*1436, DRB1*1437, DRB1*1438, DRB1*1439, DRB1*1440, DRB1*1441, DRB1*1442, DRB1*1443, DRB1*1444, DRB1*1445, DRB1*1446, DRB1*1447, DRB1*1448, DRB1*150101, DRB1*150102, DRB1*150103, DRB1*150104, DRB1*150105, DRB1*150201, DRB1*150202, DRB1*150203, DRB1*1503, DRB1*1504, DRB1*1505, DRB1*1506, DRB1*1507, DRB1*1508, DRB1*1509, DRB1*1510, DRB1*1511, DRB1*1512, DRB1*1513, DRB1*1514, DRB1*1515, DRB1*160101, DRB1*160102, DRB1*160201, DRB1*160202, DRB1*1603, DRB1*1604, DRB1*160501, DRB1*160502, DRB1*1607, DRB1*1608, DRB2*0101, DRB3*010101, DRB3*01010201, DRB3*01010202, DRB3*010103, DRB3*010104, DRB3*0102, DRB3*0103, DRB3*0104, DRB3*0105, DRB3*0106, DRB3*0107, DRB3*0108, DRB3*0109, DRB3*0110, DRB3*0111, DRB3*0201, DRB3*020201, DRB3*020202, DRB3*020203, DRB3*020204, DRB3*0203, DRB3*0204, DRB3*0205, DRB3*0206, DRB3*0207, DRB3*0208, DRB3*0209, DRB3*0210, DRB3*0211, DRB3*0212, DRB3*0213, DRB3*0214, DRB3*0215, DRB3*0216, DRB3*0217, DRB3*0218, DRB3*0219, DRB3*030101, DRB3*030102, DRB3*0302, DRB3*0303, DRB4*01010101, DRB4*0102, DRB4*01030101, DRB4*01030102N, DRB4*010302, DRB4*010303, DRB4*010304, DRB4*0104, DRB4*0105, DRB4*0106, DRB4*0107, DRB4*0201N, DRB4*0301N, DRB5*010101, DRB5*010102, DRB5*0102, DRB5*0103, DRB5*0104, DRB5*0105, DRB5*0106, DRB5*0107, DRB5*0108N, DRB5*0109, DRB5*0110N, DRB5*0111, DRB5*0112, DRB5*0113, DRB5*0202, DRB5*0203, DRB5*0204, DRB5*0205, DRB6*0101, DRB6*0201, DRB6*0202, DRB7*010101, DRB7*010102, DRB8*0101, and DRB9*0101.

A subsequence length of 25 nucleotides was selected, and maximal sequential overlap was used to provide the series of subsequences. The second exon was chosen as the starting point for the analysis, with the first 25-mer subsequence positioned such that the 13^(th) nucleotide of the subsequence (underlined, see below) aligned with the first base of the second exon. This is shown below using a reference sequence typical of many DRB alleles as follows:

intron 1    exon 2        . . . GTGTCCCCACAGCACGTTTCTTGTG . . .

Step 1: Defining Subsequences for Selecting Probes Centered on the First Nucleotide of the Second Exon.

The first subject subsequence is the 25 nucleotide subsequence of the DRB locus about the interface of intron 1 and exon 2. This first subsequence is generated against the first nucleotide in exon 1 (the underlined “C” residue): GTGTCCCCACAGCACGTTTCTTGTG (this sequence is a reference sequence found in 26 alleles).

Step 2: Defining Subsequences for Selecting Probes Centered on the Second Nucleotide of the Second Exon.

The protocol of step 1 is repeated, except that 25-mer subsequence is centered on the second nucleotide. Again, considering a reference sequence the 25-mer is: TGTCCCCACAGCACGTTTCTTGTGG.

Steps 3 to 284. Defining Subsequences for Selecting Probes Centered on the 3^(rd) to 284^(th) Nucleotide of the Second Exon.

The protocol of step 1 is repeated for each nucleotide in the exon.

Step 285: Pooling of 25-mer Subsequences

All 25-mer subsequences for each allele of the locus are combined to form a set of target nucleotide sequences capable of identifying all alleles of the locus.

Step 286: Removal of Redundant Subsequences

All subsequences are analyzed, and redundant sequences (exact matches) are removed to leave only unique subsequences. It is estimated that if the process was carried out for all 270 nucleotides of the second exon, only about 5,500 unique subsequences would be generated. This is a significant reduction in probe number predicted in the prior art.

EXAMPLE 2 Production of Microarray Chip

The 5,500 target nucleotide sequences in the pool are synthesized directly onto a microarray chip by Affymetrix Inc who provide a custom gene chip array service.

EXAMPLE 3 Use of Probes to Assign Identify DRB Allele for an Individual Patient Sample.

DNA extraction of peripheral blood or buccal smear is standard practice. Approx. 1,000 ng of DNA is recommended for microarray assay.

Long PCR.

Primers can be located in introns, exons or a combination. For instance, for HLA-DRB typing, primers are selected upstream in intron 1, and downstream in exon 6. The amplicon is approx. 5.1 kb. The disadvantage of using intron sequences as primer sites is that there is usually less sequence data, and absence of data corresponding to exon alleles, than for corresponding exon sequence. For HLA-DRB, published data provides sufficient intron 1 data for primer selection. However, even in this case, further sequencing is near certain to reveal new SNPs. If they occur in the primer sequence, it can be expected to complicate amplification of sequences bearing that new variant. The alternative is to utilise exon sequences since these have been more extensively studied. For HLA-DRB there are sites suitable as primers further upstream, in exon 1, Since amplicons using exon 1 and exon 6 primers span the full length of the 8 kb intron 1, the resulting amplicon is over 13 kb in length. Applicants have confirmed the suitability of the commercial Long PCR kit for amplification of 17 kb, so the exon only primered amplicon is also suitable.

Fragmentation of Amplicons.

The protocol process is non-specific, resulting in the shearing of the amplicons into fragments of tens to low hundreds of nucleotides required for efficient hybridisation to the chip-adherent probes. Details provided in the following document GeneChip® CustomSeq™ Resequencing (Array Protocol) Version 2.0, 701231 Rev. 3; the entire contents of which is incorporated by reference. This document can be obtained from Affymetrix Inc (Technical Support) 3380 Central Expressway Santa Clara, Calif. 95051 U.S.A.

Hybridisation.

Details are provided in GeneChip® CustomSeq™ Resequencing (Array Protocol) Version 2.0, 701231 Rev. 3

Allele Assignment.

Allele assignment is achieved by relating the probe hybridisation patterns to allele sequence variation by an iterative reduction algorithm (Helmberg W, Lanzer G, Zahn R, Weinmayr B, Wagner T, Albert E. Virtual DNA analysis—a new tool for combination and standardised evaluation of SSO, SSP and sequencing-based typing results. Tissue Antigens. 1998 June; 51(6):587-92.)

EXAMPLE 4 Generation of Probe Set for Assignment of Allele Types at HLA-A*0201 (Exons 2 and 3)

The following exon sequences of HLA*0201 were used to generate a probe set for assignment of HLA-A*0201. For the purposes of probe generation, the exon sequences were extended by 12 nucleotides in both 5′ and 3′ directions into the adjacent intronic regions.

Exon2: GCTCCCACTCCATGAGGTATTTCTTCACATCCGTGTCCCGGCCCGGCCGC GGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGT GCGGTTCGACAGCGACGCCGCGAGCCAGAGGATGGAGCCGCGGGCGCCGT GGATAGAGCAGGAGGGTCCGGAGTATTGGGACGGGGAGACACGGAAAGTG AAGGCCCACTCACAGACTCACCGAGTGGACCTGGGGACCCTGCGCGGCTA CTACAACCAGAGCGAGGCCG Exon 3 GTTCTCACACCGTCCAGAGGATGTATGGCTGCGACGTGGGGTCGGACTGG CGCTTCCTCCGCGGGTACCACCAGTACGCCTACGACGGCAAGGATTACAT CGCCCTGAAAGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTC AGACCACCAAGCACAAGTGGGAGGCGGCCCATGTGGCGGAGCAGTTGAGA GCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGATACCTGGAGAA CGGGAAGGAGACGCTGCAGCGCACGG

A subsequence length of 25 was chosen, and maximum overlap utilized.

Probe sets that are capable of identifying the above hypervariable Exon 2/3 regions are shown in FIG. 2. Where it is desired to identify hypervariable regions other than that shown above, the probe generation process is repeated for each hypervariable region. The redundant probe sequences may then be removed.

Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein. 

1. A method for identifying a set of target nucleotide sequences capable of identifying a member of a group of related nucleotide sequences, the method comprising the step of dividing the nucleotide sequence of each member of the group into a plurality of subsequences, wherein at least two of the subsequences overlap.
 2. A method according to claim 1 wherein at least three of the subsequences overlap with each other.
 3. A method according to claim 1 wherein at least four of the subsequences overlap with each other.
 4. A method according to claim 1 wherein at least five of the subsequences overlap with each other.
 5. A method according to claim 1 wherein the overlap is complete overlap.
 6. A method according to claim 1 comprising the step of analyzing at least a portion of the subsequences for redundancy.
 7. A method according to claim 1 wherein one or more of the subsequences does not contain one or more polymorphic sites at, or toward, the 5′ and/or 3′ ends of the one or more subsequences.
 8. A method according to claim 1 wherein one or more of the subsequences contains one or more polymorphic sites at, or toward, the center of the one or more subsequences.
 9. A method according to claim 1 wherein one or more of the subsequences contain one polymorphic site at the center of the one or more subsequences.
 10. A method according to claim 1 wherein the related sequences differ by the presence of one or more nucleotide polymorphisms.
 11. A method according to claim 10 wherein the nucleotide polymorphisms are single nucleotide polymorphisms.
 12. A method according to claim 1 wherein the subsequences are probe-length.
 13. A method according to claim 1 wherein the subsequences are from about 10 to about 50 nucleotides in length.
 14. A method according to claim 1 wherein the subsequences are from about 15 to about 35 nucleotides in length.
 15. A method according to claim 1 wherein the subsequences are about 25 nucleotides in length.
 16. A method according to claim 1 wherein all subsequences are of the same or similar length.
 17. A method according to claim 1 wherein the related nucleotide sequences have a sequence identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99%.
 18. A method according to claim 1 wherein the related sequences exhibit SNPs at a high density.
 19. A method according to claim 1 wherein the related sequences are protein coding, non-coding, or a combination of protein coding and non-coding.
 20. A method according to claim 1 wherein the related sequences are directed to the same region of a genome.
 21. A method according to claim 1 wherein the related nucleotide sequences are alleles of a gene.
 22. A method according to claim 1 wherein the number of related nucleotide sequences in the group of related nucleotide sequences is more than 100, 200, 300, 400, 500, 600, 700, 800, 900 or
 1000. 23. A method according to claim 1 wherein the related nucleotide sequences are part of a gene locus involved in the immune system.
 24. A method according to claim 23 wherein the locus is a locus of the Major Histocompatability Complex (MHC), the T-cell receptor, the B-cell receptor, the Killer Inhibitory Receptor, or an immunoglobulin.
 25. A method according to claim 23 wherein the locus is a locus of the Human Leukocyte Antigen (HLA) system.
 26. A method according to claim 23 wherein the wherein the locus is a Class I or Class II MHC transmembrane protein.
 27. A method according to claim 23 wherein the locus is a DR, DQ or DP locus.
 28. A method according to claim 6 comprising removal or non-inclusion of at least one redundant sequence from the set of target nucleotide sequences.
 29. A method according to claim 28 wherein the method reduces the number of sequences in the set of target nucleotide sequences by a multiple of at least about 5, 10 or 20 from the number of probes expected by theory.
 30. A method according to claim 28 wherein the method reduces the probe number by at least about 50%, 60%, 70%, 80%, 90% or 95%.
 31. A method according to claim 28 wherein substantially all redundant sequences are removed, or are not included, in the probe set.
 32. A method according to claim 1, wherein the method is amenable to automation.
 33. A method according to claim 1, wherein the method is capable of identifying new polymorphic sites, or new combinations of polymorphic sites in the related sequences.
 34. A probe set capable of specifically hybridizing to target nucleotide sequences identified by a method according to claim
 1. 35. A probe set according to claim 34 wherein at least one probe comprises a label selected from the group consisting of Cy5, Cy3, FITC, rhodamine, biotin, DIG and a radioisotope.
 36. A solid matrix including an immobilized probe set according to claim
 34. 37. A solid matrix according to claim 36, wherein the solid matrix is a microarray chip.
 38. A method of identifying a member of a group of related nucleotide sequences using a probe set according to claim
 34. 39. A method according to claim 38 comprising the step of recovering a member of a group of related nucleotide sequences using a probe set according to claim
 34. 40. A method according to claim 39 comprising the steps of exposing the probe set to the group of related nucleotide sequences under conditions allowing a probe of the probe set to bind to a nucleotide sequence of the group of related nucleotide sequences to form a probe/nucleotide sequence complex, and substantially isolating the probe/nucleotide sequence complex.
 41. A method of definitive allele assignment comprising use of a probe set according to claim
 34. 42. A method of transplantation tissue typing based on the HLA system comprising use of a probe set according to claim
 34. 43. A method of identifying a new allele comprising use of a probe set according to claim
 34. 44. A computer executable code capable of executing a method according to claim
 1. 45. A method according to claim 1 substantially as herein before described with reference to any of the Figures or Examples. 